Abstract
Both the 5' cap (m7GpppN) and the 3' poly(A) tail of eukaryotic mRNAs are important regulators of translation efficiency in vivo. Their function, however, is markedly reduced in in vitro translation systems derived from either rabbit reticulocytes or wheat germ. The impact of exogenous poly(A) on cap-dependent translation was examined in vitro. The translation of uncapped mRNA was preferentially repressed in the presence of exogenous poly(A). As a result, translation became increasingly cap-dependent with the increase in exogenous poly(A). The translation in wheat germ lysate was stimulated by the addition of purified eukaryotic initiation factor (eIF)-4B or eIF-4F; however, addition of poly(A) prevented this eIF-4B- or eIF-4F-mediated stimulation. Addition of eIF-4F or eIF-4B, alone, was not sufficient to restore translation in lysate to which poly(A) had been added. Restoration, however, was observed when eIF-4F, eIF-4B, and eIF-4A were added in combination. These data suggest that 1) exogenous poly(A) may bind to and sequester factors required for translation and 2) that capped messages compete with poly(A) more efficiently for these factors than do uncapped mRNAs. Gel shift analysis of purified initiation factors isolated from wheat germ confirmed that eIF-4B and eIF-4F do in fact form complexes with poly(A) in vitro.
Highlights
Both the 5' cap (m'GpppN) and the 3' poly(A) tail of lysates has not yet been possible
An excess of factors infrom either rabbit reticulocytes or wheat germT. he im- volved in initiation may reduce the requirement for a cap or pact of exogenous poly(Ao)n cap-dependent translation poly(A) tail
Previous studies have disagreeodn the effect that exogenous mRNA was preferentially repressed in the presence of exogenous poly(A).As a result, translation became increasinglycap-dependentwith the increase in exogenous poly(A).Translation in wheatgerm lysate was stimulated bythe addition of purified eukaryoticnitiationfactor(eIF)-4BoreIF-4F;,additionof poly(Ap)revented this eIF-4Bo-erIF-4F-mediated poly(A)has on translation in lysate(sLodish and Nathan,1972; JacobsonandFavreau, 1983; Munroeand Jacobson,1990b; Bablanian et aZ., 1991;Grossi de Sa et al, 1988)
Summary
Wheat germ thanin vivo (Gallie, 1991). the effect mRNA Constructs and in Vitro fianscription Reaction Conditionsof a cap on stimulating translation irseduced by approximately two orders of magnitude in vitro compared t o the effect in plant and animalcells (Gallie, 1991).As a result, a biochemical analysis of the synergism between a cap and a poly(A) tail using. (Yisraeli and Melton,1989)using 40 mM Tris-HC1 pH 7.5,6 mM MgCl,, 2 mM spermidine,100pg/ml bovine serum albumin, 0.5 m~ each ofATP, CTP, UTP, plus 160 p~ GTP, 1mM m'GpppG, 100 mM dithiothreitol,0.3 l The abbreviations used are: eIF, eukaryotic initiation factor;Tricine, N-[2-hydroxy-l,l-bis(hydroxymethyl)ethyllglycineT;MVt,obacco mosaic virus; TEV, tobacco etch virus. Gel Retardation-5 ng of labeled RNA and either purified eIFs or 20 pg of crude extract were usfeodr the binding reactions i1n4-1a.11volume containing 10mM Tris, pH 7.5, 35 mM KCl, 1.0 mM MgCl,, 5% glycerol, 1mM dithiothreitol, 0.7 mg/ml total yeastRNA, 0.5 uniffpl RNasin.
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